Our recent studies have shown that the cardiac contractile responses to both beta1 adrenergic receptor (beta1AR) and beta2AR stimulation are markedly decreased with aging in rat ventricular myocytes. However, the cellular and molecular mechanisms underlying the age-associated changes are not well understood. In this study we examined possible mechanisms for the age-associated changes in betaAR subtype responsiveness. The present results have demonstrated that these age-associated depressions in betaAR subtype contractile responses are accompanied by significant reductions in both beta1AR and beta2AR densities and adenylyl cyclase activity. Pertussis toxin, PTX, treatment specifically potentiates the positive inotropic effect of beta2AR, but not beta1AR stimulation in both young and old groups (2 and 24 mo). However, the age-associated diminutions in both betaAR subtype inotropic effects are not reversed by the PTX treatment, suggesting that the age-associated changes are not caused by an up-regulation of PTX-sensitive G proteins with aging. This is further confirmed by the observation that the level of Gi protein is not altered with aging. In addition, the messenger RNA (mRNA) levels and enzymatic activity of G protein-coupled receptor kinase5 (GRK5) and beta-adrenergic receptor kinase (beta ARK) do not change with aging, suggesting that neither beta ARK nor betaGRK5 appears to be involved in the age- associated diminution of either beta1AR or beta2AR response. Therefore, we conclude that both cardiac beta 1AR and beta 2AR densities are significantly affected by aging, which may, at least in part, contribute to the age-related deficit in the inotropic effects of both betaAR subtype stimulation. The decreased adenylyl cyclase activity may also play an important role in the reduced beta1AR but not beta2AR response in aged hearts, since the inotropic effect of beta 2AR stimulation is dissociated from cAMP production. Further studies are required to determine whether mechanisms other than those examined in the present study, e.g., changes in Gs proteins or effectors, play a role in the decreased contractile responses to both beta1AR and beta2AR stimulation with aging.